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Ethnopharmacological Relevance: Phytolacca acinosa Roxb. (PAR) is a Traditional Chinese Medicinal (TCM) plant with a broad global distribution encompassing 35 species, four of which are found in the People's Republic of China. It occupies a significant role in both Oriental and American traditional medicine, employed in treating a range of conditions such as edema, inflammation, dermatitis, and rheumatism. PAR is also used as a molluscicide and for addressing tumors and bronchitis. The plant is documented in the Chinese Pharmacopoeia and has a longstanding history in TCM, particularly for its diuretic properties and in treating ailments such as edema, swelling, and ulcers. Notably, PAR has demonstrated potent inhibitory effects against the A549 human lung cancer cell line, underscoring its potential in contributing to the development of novel cancer therapeutics., Aim of the Study: The research aims to elucidate the active components of PAR and their mechanisms in treating hepatocellular carcinoma (HCC)., Materials and Methods: Employing network pharmacology, this study predicted the principal active compounds and key targets of PAR. A holistic methodology incorporating biological network analysis, transcriptomics sequencing, molecular docking, and molecular dynamics (MD) simulations was utilized to forecast the effects of PAR on HCC, with empirical evidence supporting these findings., Results: Network pharmacology identified xanthomicrol as the foremost active compound in PAR. The tumor-suppressive functions of PAR, as indicated by KEGG pathway analysis and transcriptomics sequencing, predominantly occur via the PI3K/AKT pathway. Molecular docking and dynamics simulations demonstrated the high affinity of xanthomicrol towards TNF, MMP9, PPARG, KDR, and MMP2. In vivo experiments verified the efficacy of xanthomicrol in curtailing HCC tumor growth, while in vitro assessments revealed its substantial impact on the proliferation and apoptosis of HepG2 and HCCLM3 cells. Moreover, the study indicates that xanthomicrol may modulate the expression of TNF, MMP9, PPARG, KDR, and MMP2 in HCC cells and inhibit the activation of the PI3K/AKT pathway., Conclusions: Xanthomicrol, a principal active component of PAR, has been identified to impede the growth of HCC by targeting the PI3K/Akt/MMP9 pathway. This insight could enhance therapeutic approaches for HCC., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024. Published by Elsevier B.V.)

In China, the dry roots of Phytolacca acinosa or P. americana in the Phytolacca plants are listed as the traditional Chinese medicine(TCM) Phytolaccae Radix. Phytolacca plants contain triterpenoids, triterpenoid saponins, polysaccharides, antiviral proteins, flavonoids, and other chemical components. The most important compounds are triterpenoids and triterpenoid saponins.Phytolacca plants have anti-inflammatory, anti-tumor, antibacterial, antifungal, antimalarial, deworming, and other activities. In this paper, the chemical constituents, pharmacological effects, toxicity, and heavy metal enrichment characteristics of Phytolacca plants were summarized to provide a theoretical basis for the study of active ingredients and drug development of Phytolacca plants.

Background: Extensive antifungal drug use has enhanced fungal resistance, resulting in persistent mycoses. Combining antifungal plant extracts/compounds with these drugs offers good alternatives to increase the activity of both partners, minimize side effects, and overcome drug resistance. In our previous study, Phytolacca tetramera berries extracts demonstrated activity against Candida spp., correlating with the amount of the main constituent phytolaccoside B and its genin, phytolaccagenin. The extracts and phytolaccagenin altered the fungal plasma membrane by binding to ergosterol, whereas phytolaccoside B increased chitin synthase activity. However, the presence of triterpenoid saponins in Phytolacca spp. has been linked to acute toxicity in humans., Purpose: This study aimed to evaluate combinations of P. tetramera berries extracts, phytolaccoside B and phytolaccagenin, together with commercial antifungals [amphotericin B, fluconazole, itraconazole, posaconazole, and caspofungin] against Candida albicans and Candida glabrata, to find synergistic effects with multi-target actions, in which the doses of both partners are reduced, and therefore their toxicity. Additionally, we intended to explore their anti-virulence capacity, thereby hindering the development of drug-resistant strains., Methods: The effects of these combinations were evaluated using both the checkerboard and isobologram methods. Fractional Inhibitory Concentration Index and Dose Reduction Index were calculated to interpret the combination results. To confirm the multi-target effect, studies on mechanisms of action of synergistic mixtures were performed using ergosterol-binding and quantification assays. The ability to inhibit Candida virulence factors, including biofilm formation and eradication from inert surfaces, was also evaluated. Quantification of active markers was performed using a validated UHPLC-ESI-MS method., Results: Eight synergistic combinations of P. tetramera extracts or phytolaccagenin (but not phytolaccoside B) with itraconazole or posaconazole were obtained against C. albicans, including a resistant strain. These mixtures acted by binding to ergosterol, decreasing its whole content, and inhibiting Candida biofilm formation in 96-well microplates and feeding tubes in vitro, but were unable to eradicate preformed biofilms., Conclusions: This study demonstrated the synergistic and anti-virulence effects of P. tetramera berries extracts and phytolaccagenin with antifungal drugs against Candida spp., providing novel treatment avenues for fungal infections with reduced doses of both natural products and commercial antifungals, thereby mitigating potential human toxicity concerns., Competing Interests: Declaration of competing interest The authors declare no conflicts of interest., (Copyright © 2024 Elsevier GmbH. All rights reserved.)

Phytolacca dodecandra (L' Herit), or 'Endod', is one of the widely known medicinal plants in Ethiopia. Berries of the endod have been used as a detergent for centuries. The present study was aimed to test the hepatoprotective effects of the plant against acetaminophen (APAP)-induced liver injury in rats. Mice of either sex were used for oral acute toxicity tests and APAP-induced lethality tests. Hepatoprotective experiments were done on male rats using 2 g/kg of APAP to induce liver damage. Liver enzymes, total bilirubin (TB), and lipid profile were determined. Liver tissues were also examined histopathologically to see a morphologic change in the control and experiment groups. The protective effect of the plant extract was also tested through sodium pentobarbital (SPB)-induced sleeping time. A significant increase in serum levels of liver enzymes, TB, low-density lipoprotein (LDL), and triglycerides (TGs) was seen from oral administration of 2 g/kg APAP. Total cholesterol (TC) and high-density lipoprotein (HDL) levels were decreased. Serum levels of all parameters were reversed to normal after administration of silymarin 100 mg/kg and, 100, 200, and 400 mg/kg doses of the extract. A significant dose-dependent hepatoprotective effect of Phytolacca dodecandra Methanol Root Extract (PDME) was seen in terms of LDL. Histopathological investigations and SPB-induced sleeping time confirmed the findings of biochemical analysis. The findings of the present study indicate that PDME protected the liver from APAP injury., Competing Interests: Declaration of Conflicting InterestsThe authors declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.

Two new oleanane-type triterpene glycosides, phytolasides A ( 1 ) and B ( 2 ), and six known ones ( 3 - 8 ), were isolated from Phytolacca acinosa fruit fermentation broth. Their structures were elucidated by HR-ESI-MS and 1 D- and 2 D-NMR spectroscopic methods. Antiproliferation of compounds 1 and 2 against HepG2 cells was examined by using CCK8 assays.

Triterpene saponins (TSs) are important bioactive constituents with structural diversity widely distributed in many plants. The root of Phytolacca acinosa Roxb (RPa) has been used as a traditional Chinese medicine. However, TSs as the main active ingredients in RPa have not been fully characterized. Here, we profiled TSs from RPa by high-performance liquid chromatography coupled to electrospray ionization and quadrupole time-of-flight mass spectrometry (HPLC-ESI-QTOF-MS/MS). We tentatively identified 29 TSs, including 13 that had not been reported previously from this plant. This study indicates that HPLC-ESI-QTOF-MS/MS is an effective and rapid method for the characterization of complicated TSs in herbal extracts., (© The Author(s) 2021. Published by Oxford University Press. All rights reserved. For permissions, please email: journals.permissions@oup.com.)

Background: Esculentosides and related phytolaccosides form a group of oleanene-type saponins isolated from plants of the Phytolaccaceae family, essentially Phytolacca esculenta, P. americana and P. acinosa. This chemical family offers a diversity of glycosylated compounds, including molecules with a mono-, di- or tri-saccharide unit at position C-3, and with or without a glucose residue at position C-28. The esculentosides, which derive essentially from the sapogenin jaligonic acid or its 30-methyl ester phytolaccagenin, exhibit anti-inflammatory, antifungal and anticancer activities., Purpose: The objective of the review was to identify the 26 esculentosides (ES) and phytolaccosides known to date, including 16 monodesmosidic and 10 bidesmosidic saponins, and to review their pharmacological properties and molecular targets., Methodology: The retrieval of potentially relevant studies was done by systematically searching of scientific databases like Google Scholar and PubMed in January-May 2020. The main keywords used as search terms were related to esculentosides, phytolaccosides and Phytolaccaceae. The systematic search retrieved about 110 papers that were potentially relevant and after an abstract-based selection, 68 studies were analyzed in details and discussed., Results: The structural relationship between the compounds and their sapogenin precursors has been studied. In addition, the pharmacological properties of the main ES, such as ES-A, -B and -H, have been analyzed to highlight their mode of action and potential targets. ES-A is a potent inhibitor of the release of cytokines and this anti-inflammatory activity contributes to the anticancer effects observed in vitro and in vivo. Potential molecular targets of ES-A/B include the enzymes cyclooxygenase 2 (COX-2) and casein kinase 2 (CK2). In addition, the targeting of the protein high-mobility group box 1 (HGMB1) by ES-A/B is proposed, based on molecular modeling and the structural analogy with the related saponin glycyrrhizin, a potent HGMB1 alarmin inhibitor., Conclusion: More work is needed to properly characterize the molecular targets but otherwise compounds like ES-A and ES-H emerge as potent anti-inflammatory and anticancer agents and ES-B as an antifungal agent. A preclinical development of these three compounds should be considered., (Copyright © 2020 Elsevier GmbH. All rights reserved.)

Using the pathosystem Phaseolus vulgaris -tobacco necrosis virus (TNV), we demonstrated that PD-L1 and PD-L4, type-1 ribosome inactivating proteins (RIPs) from leaves of Phytolacca dioica L., possess a strong antiviral activity. This activity was exerted both when the RIPs and the virus were inoculated together in the same leaf and when they were inoculated or applied separately in the adaxial and abaxial leaf surfaces. This suggests that virus inhibition would mainly occur inside plant cells at the onset of infection. Histochemical studies showed that both PD-L1 and PD-L4 were not able to induce oxidative burst and cell death in treated leaves, which were instead elicited by inoculation of the virus alone. Furthermore, when RIPs and TNV were inoculated together, no sign of H 2 O 2 deposits and cell death were detectable, indicating that the virus could have been inactivated in a very early stage of infection, before the elicitation of a hypersensitivity reaction. In conclusion, the strong antiviral activity is likely exerted inside host cells as soon the virus disassembles to start translation of the viral genome. This activity is likely directed towards both viral and ribosomal RNA, explaining the almost complete abolition of infection when virus and RIP enter together into the cells.

Phytolacca acinosa is an herb for treatment of ascites and tumor. Two forms of P. acinosa, i.e. raw and vinegar-processed herb, have been used in clinic. However, pharmacokinetic difference between the two forms of P. acinosa has not been fully understood. Herein, a comparative pharmacokinetic method based on liquid chromatography with tandem mass spectrometry was developed for quantification of six bioactive triterpenoids, including esculentoside H, esculentoside T, esculentoside A, esculentoside B, phytolaccagenic acid, and phytolaccagenin in rat plasma after oral administration of different forms of P. acinosa. Separation was performed on an Acquity BEH C 18 column (1.7 µm, 2.1 mm × 50 mm). The method was validated over a linear range of 2.0-5000 ng/mL. Intraday and interday bias were within ±5%. Besides, all triterpenoids were stable in plasma during different storage conditions. The described method was successfully applied to a comparative pharmacokinetic study of raw and vinegar-processed P. acinosa in rats. Notably, double peak phenomenon for six triterpenoids of P. acinosa was observed for the first time. AUC 0→t and C max values of esculentoside H, esculentoside T, phytolaccagenic acid, and phytolaccagenin were significantly lower in vinegar-processed group than that of raw group, indicating the oral bioavailability of the four triterpenoids was decreased after vinegar processing., (© 2020 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Rare earth elements (REEs) are now considered emerging pollutants in the environment. Phytolacca americana, an REE hyperaccumulating plant, has been proposed for the remediation of REE-contaminated soils. However, there is no REE-related information for other Phytolacca species. Here, we examined five species (P. americana, P. acinosa, P. clavigera, P. bogotensis, and P. icosandra) for their response to REEs. REE accumulation and fractionation traits both occurred on the same order of magnitude among the five species. Heavy REEs were preferentially transferred to leaves relative to light REEs. Regardless of the species, lateral root length and chlorophyll content decreased under REE exposure, and lateral roots and foliar anthocyanins increased. However, plants did not experience or only slightly experienced oxidative stress. Finally, REE exposure strongly modulated the ionome of roots and, to a lesser extent, that of leaves, with a negative correlation between REE and Mn contents. In conclusion, our study provides new data on the response of several Phytolacca species to REEs. Moreover, we highlighted that the REE accumulation trait was conserved among Phytolacca species. Thus, we provide valuable information for the phytoremediation of REE-contaminated sites since the most appropriate Phytolacca species could be selected depending on the climatic/pedological area to be remediated.

Phytolacca, which belongs to the family of Phytolaccaceae, are known for their use in popular medicine. Bioactivity of five extracts from Phytolacca dioica seeds were evaluated in four bioassays. A selected group of compounds from the extract that displayed the best bioactivity was analysed. The ethyl acetate extract (EAE) possessed the highest content of phenolics, the highest inhibitory activity on the tyrosinase and xanthine oxidase enzymes and showed a high antioxidant activity. HPLC-DAD-MS was employed to identify the phenolics profile of the most active one (EAE). HSCCC analysis of the EAE led to the isolation of phytolaccoside B and a mixture of 4 isomers, isoamericanol B1, B2, C1 and C2. These isoamericanol isomers presented activity against tyrosinase and xanthine oxidase. Our results revealed for the first time an interesting biological activity of the extract and isolated compounds from P. dioica seeds, which could be considered as a source of bioactive molecules.

Background: Phytolacca tetramera is an endemic plant from Argentina that is currently at serious risk because its environment is subjected to a high anthropic impact. A previous study has shown that berry extracts obtained from this plant display antifungal activity against multiple human-pathogenic fungi when tested with a non-standardized method. Further evidences of the antifungal properties of other parts of the plant and studies of mechanism of antifungal action of the antifungal chemically characterized extracts are required., Purpose: This study aimed to gain further evidence of the antifungal activity of P. tetramera berry, leaf and root extracts in order to find the most active extract to be developed as an Herbal Medicinal Antifungal Product. The medicinal usefulness of P. tetramera extracts as antifungal agents will serve as an important support to create concience and carry out actions tending to the preservation of this threatened species and its environment., Materials and Methods: Chemical analysis of all P. tetramera extracts, including quantitation of selected markers, was performed through UHPLC-ESI-MS/MS and UPLC-ESI-MS techniques according to the European Medicines Agency (EMA). The antifungal activity of the quantified extracts was tested with the standardized CLSI microbroth dilution method against Candida spp. Antifungal mechanisms of the most active extract were studied by examination of morphological changes by phase-contrast and fluorescence microscopies and both, cellular and enzymatic assays targeting either the fungal membrane or the cell wall., Results: The antifungal activity of twelve P. tetramera extracts was tested against Candida albicans and Candida glabrata. The dichloromethane extract from berries (PtDEb) showed the best activity. Phytolaccagenin (PhytG) and phytolaccoside B (PhytB) were selected as the main active markers for the antifungal P. tetramera extracts. The quantitation of these active markers in all extracts showed that PtDEb possessed the highest amount of PhytG and PhytB. Finally, studies on the mechanism of antifungal action showed that the most active PtDEb extract produces morphological changes compatible with a damage of the cell wall and/or the plasma membrane. Cellular and enzymatic assays showed that PtDEb would not damage the fungal cell wall by itself, but would alter the plasma membrane. In agreement, PtDEb was found to bind to ergosterol, the main sterol of the fungal plasma membrane., Conclusion: Studies of the anti-Candida activity of P. tetramera extracts led to the selection of PtDEb as the most suitable extract, confirming the antifungal properties of the threatened species P. tetramera. The new data give a valuable reason for the definitive protection of this sp. and its natural environment thus allowing further studies for the future development of an Herbal Medicinal Antifungal Product., (Copyright © 2019 Elsevier GmbH. All rights reserved.)

Purpose: The present randomized controlled clinical trial evaluated the efficacy of homeopathic medicines of Melissa officinalis (MO), Phytolacca decandra (PD), and the combination of both in the treatment of possible sleep bruxism (SB) in children., Study Design: Patients (n = 52) (6.62 ± 1.79 years old) were selected based on the parents report of SB. The study comprised a crossover design that included 4 phases of 30-day treatment (Placebo; MO 12c; PD 12c; and MO 12c + PD 12c), with a wash-out period of 15 days between treatments., Methods: At baseline and after each phase, the Visual Analogic Scale (VAS) was used as the primary outcome measure to evaluate the influence of treatments on the reduction of SB. The following additional outcome measures were used: a children's sleep diary with parent's/guardian's perceptions of their children's sleep quality, the trait of anxiety scale (TAS) to identify changes in children's anxiety profile, and side effects reports. Data were analyzed by ANOVA with repeated measures followed by Post Hoc LSD test., Results: Significant reduction of SB was observed in VAS after the use of Placebo (-1.72 ± 0.29), MO (-2.36 ± 0.36), PD (-1.44 ± 0.28) and MO + PD (-2.21 ± 0.30) compared to baseline (4.91 ± 1.87). MO showed better results compared to PD (p = 0.018) and Placebo (p = 0.050), and similar result compared to MO+PD (p = 0.724). The sleep diary results and TAS results were not influenced by any of the treatments. No side effects were observed after treatments., Conclusion: MO showed promising results in the treatment of possible sleep bruxism in children, while the association of PD did not improve MO results., (Copyright © 2019. Published by Elsevier GmbH.)

Antimicrobial peptides, also called Host Defence Peptides (HDPs), are effectors of innate immune response found in all living organisms. In a previous report, we have identified by chemical fragmentation, and characterized the first cryptic antimicrobial peptide in PD-L4, a type 1 ribosome inactivating protein (RIP) from leaves of Phytolacca dioica L. We applied a recently developed bioinformatic approach to a further member of the differently expressed pool of type 1 RIPs from P. dioica (PD-L1/2), and identified two novel putative cryptic HDPs in its N-terminal domain. These two peptides, here named IKY31 and IKY23, exhibit antibacterial activities against planktonic bacterial cells and, interestingly, significant anti-biofilm properties against two Gram-negative strains. Here, we describe that PD-L1/2 derived peptides are able to induce a strong dose-dependent reduction in biofilm biomass, affect biofilm thickness and, in the case of IKY31, interfere with cell-to-cell adhesion, likely by affecting biofilm structural components. In addition to these findings, we found that both PD-L1/2 derived peptides are able to assume stable helical conformations in the presence of membrane mimicking agents (SDS and TFE) and intriguingly beta structures when incubated with extracellular bacterial wall components (LPS and alginate). Overall, the data collected in this work provide further evidence of the importance of cryptic peptides derived from type 1 RIPs in host/pathogen interactions, especially under pathophysiological conditions induced by biofilm forming bacteria. This suggests a new possible role of RIPs as precursors of antimicrobial and anti-biofilm agents, likely released upon defensive proteolytic processes, which may be involved in plant homeostasis., (Copyright © 2018 Elsevier B.V. All rights reserved.)

A new triterpenoid saponin named esculentoside U(1), along with the five known compounds, was isolated and characterized from the roots of Phytolacca acinosa, a commonly used traditional Chinese medicine with anti-inflammatory and anti-rheumatoid activities. The structure of the new saponin was elucidated as 3-O-[β-D-glucopyranosyl-(1→4)]-β-D-xylopyranosyl]-2, 23-dihydroxyolean-11, 13(18)-diene-28, 29-dioic acid 29-methyl ester(1). The assignment of all NMR signals of 1 was performed by means of 2D-NMR experiments., Competing Interests: The authors of this article and the planning committee members and staff have no relevant financial relationships with commercial interests to disclose., (Copyright© by the Chinese Pharmaceutical Association.)

Two new flavones, 6,7-methylenedioxy-4-hydroxypeltogynan-7'-one (1), cochliophilin B (2), as well as two known ones, cochliophilin A (3) and 6-methoxy-7-hydroxy flavone (4), were isolated from the ethanol extract of the root of Phytolacca acinosa Roxb. Compound 1 is a flavanol framework with one δ-lactone unit, which is rather rare in nature. The structures of the new compounds were determined on the basis of extensive spectroscopic (IR, MS, 1D- and 2D-NMR) analyses, the absolute configuration of 1 was established by comparing experimental and calculated electronic circular dichroism spectra. The structures of known compounds were fixed by comparison with literatures data. Compounds 2 and 4 showed modest inhibitory activities against BEL-7402 cell line, with IC 50 values of 28.22 and 39.16 μmol/L, respectively., (© 2017 Wiley-VHCA AG, Zurich, Switzerland.)

Esculentoside A (EsA), a saponin isolated from Phytolacca esculenta, can attenuate acute liver and lung injury. However, whether EsA has a protective effect against sepsis-induced acute kidney injury (AKI) has not been reported. In this study, EsA (2.5, 5, or 10 mg/kg) was given to rats with sepsis induced by cecal ligation and puncture (CLP). We found that EsA improved the survival of septic rats in a dose-dependent manner. In addition, EsA lowered the kidney tubular damage score and decreased blood urea nitrogen and creatinine. Moreover, EsA inhibited excessive generation of pro-inflammatory tumor necrosis factor-α, IL-1β, and IL-6 in the serum and downregulated cyclooxygenase-2 and inducible nitric oxide synthase in the renal tissues of septic rats. EsA also suppressed the production of malonaldehyde and the activity of myeloperoxidase in the septic kidney and enhanced the activity of superoxide dismutase and glutathione. The anti-inflammatory and antioxidative effects of a high dose of EsA were comparable to those of dexamethasone. Mechanically, EsA inhibited CLP-induced increases in high-mobility group box 1, Toll-like receptor-4, and myeloid differentiation primary response 88 and nuclear accumulation of nuclear factor kappa B p65 in renal tissues. In vitro, lipopolysaccharide-induced alteration of AKI-related factors in HK-2 cells, which had been evaluated in vivo, was inhibited after EsA administration. Taken together, our study suggests that EsA effectively protects rats against septic AKI caused by CLP.

In this work, an innovative approach using biochar technology for hyperaccumulator disposal was developed and evaluated. The heavy metal enriched P. acinosa biomass (PBM) was pyrolyzed to produce biochar (PBC). Both PBM and PBC were characterized with X-ray diffraction (XRD) for crystal phases, scanning electron microscopy (SEM) for surface topography, and analyzed for elemental composition and mobility. The results revealed that whewellite, a dominant crystal form in biomass, was decomposed to calcite after pyrolysis. Elemental analysis indicated that 91-99% total non-volatile elements in the biomass were retained in the biochar. The toxicity characteristic leaching procedure (TCLP) results revealed that 94.6% and 0.15% of total Mn was extracted for biomass and biochar, respectively. This suggests that mobility and bioavailability of Mn in biochar was much lower relative to pristine biomass. Batch sorption experiment showed that excellent removal of aqueous silver, lead, cadmium, and copper ions can be achieved with PBC. Findings from this work indicated that biochar technology can provide a value-added solution for hyperaccumulator disposal., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

Background: The species from the genus Phytolacca constitute one of the best sources of ribosome-inactivating proteins (RIPs) that have been used both in the therapy against virus and tumors and in the construction of transgenic plants resistant to virus, bacteria, fungi and insects. Here we investigate new activities of three representative RIPs from Phytolacca dioica (dioicin 2, PD-S2 and PD-L4)., Results: The three RIPs displayed, in addition to already reported activities, rRNA N-glycosylase activities against plant, bacterial and fungal ribosomes. Additionally dioicin 2 and PD-L4 displayed endonuclease activity on a supercoiled plasmid DNA, and dioicin 2 and PD-S2 arrested the growth of the fungus Penicillium digitatum. Furthermore, dioicin 2 induced caspase activation and apoptosis in cell cultures., Conclusions: The different activities of the RIPs from Phytolacca dioica may explain the antipathogenic properties attributed to these RIPs in plants and their antiviral and antitumoral effects. In spite of the similarity in their rRNA N-glycosylase and DNA polynucleotide:adenosine glycosylase activities, they differed in their activities against viral RNA, plasmid DNA, fungi and animal cultured cells. This suggests that the presence of isoforms might optimize the response of the plant against several types of pathogens., General Significance: RIPs from Phytolacca can induce plant resistance or tumor cell death not only by means of ribosome inactivation but also by the activities found in this report. Furthermore, the induction of cell death by different mechanisms turns these RIPs into more useful tools for cancer treatment rendering the selection of RIP-resistant mutants impossible., (Copyright © 2016 Elsevier B.V. All rights reserved.)

To research the intestinal toxicity of n-BuOH fraction in Phytolacca Radix before and after being processed with vinegar. Toxic n-BuOH fractions were separated from Phytolacca Radix. In the animal model, the level of intestinal edema, water content of intestine and stool, IC₅₀ values of HT-29 and IEC-6 were detected with MTT method to compare the changes in toxicity of n-BuOH fractions from Phytolacca Radix before and after being processed with vinegar. n-BuOH fractions of Phytolacca Radix could cause intestinal edema in mice, increase the edema of duodenum, jejunum and the water content in stool, inhibit the proliferation of HT-29 cells and IEC-6 cells, indicating its intestinal toxicity, with HT-29 IC₅₀ at 14.59 mg•L⁻¹ and IEC-6 IC₅₀ at 43.77 mg•L⁻¹. After being processed with vinegar, the level of intestinal edema, edema of duodenum and jejunum and the water content in stool and inhibition ratio of cells line were reduced, with HT-29 IC₅₀ at 58.51 mg•L⁻¹ and IEC-6 IC₅₀ at 84.37 mg•L⁻¹. After being processed with vinegar, the toxicity of n-BuOH fractions from Phytolacca Radix decreased obviously., Competing Interests: The authors of this article and the planning committee members and staff have no relevant financial relationships with commercial interests to disclose., (Copyright© by the Chinese Pharmaceutical Association.)

Besides the known compounds ( ± ) 3,3-bis-demethylpinoresinol (2), americanol A (3), spergulagenic acid (4), epi-acetylaleuritolic acid (5), 6'-palmityl-α-spinateryl-d-glucoside (6a) and 6'-palmityl-δ(7)-stigmastenyl-d-glucoside (6b), a novel peltogynoid (1) named icosandrin was obtained from the dried fruits of Phytolacca icosandra. This new compound was characterised by 1D-/2D-NMR, UV, IR and HR-MS techniques as 11ξ-methoxy-6,7-methylenedioxy-[2]benzopyrano-[4,3-b][1]-benzopyran-4-one. Toxicity of 1 was assessed through the Brine Shrimp Lethality Assay. Lignan 2 is reported for the first time in Phytolaccaceae family.

In allelopathy, one plant suppresses the growth and development of other plant/plants by negatively affecting a variety of physiological and biochemical reactions. We checked the effects of methanolic extracts (allelochemical extracts) of Phytolacca latbenia (Moq.) H. Walter on antioxidant enzyme activities such as peroxidases (PODs), super oxide dismutases (SODs) and catalase (CAT) and on total protein contents (TPC), cellular injury (CI), and malondialdehyde (MDA) in the germinating seeds of Brassica napus L. (dicot) and Triticum aestivum L. (monocot). Both the crude methanolic extract root (CMER) and crude methanolic extract aerial (CMEA) of P. latbenia at 10000 ppm significantly reduced the POD activity in both the test seeds. The activity of SODs was significantly decreased by both CMER and CMEA in B. napus germinating seeds. A linear increase in the activity of CAT, CI, and MDA contents was found in both the test seeds with the increasing concentrations of CMEA and CMER, while TPC of the germinating seeds was found decreased. It is inferred that both the CMEA and CMER inhibited/delayed the seed germination, reduced the seedling growth by affecting a variety of biochemical and physiological attributes, and also caused cellular membrane injury in the germinating seeds of both the monocot and dicot seeds., (© The Author(s) 2013.)

We investigated the antimicrobial activity of PD-L4, a type 1 RIP from Phytolacca dioica. We found that this protein is active on different bacterial strains both in a native and denatured/alkylated form and that this biological activity is related to a cryptic peptide, named PDL440-65, identified by chemical fragmentation. This peptide showed the same antimicrobial activity of full-length protein and possessed, similarly to several antimicrobial peptides, an immunomodulatory effect on human cells. It assumes an alpha-helical conformation when interact with mimic membrane agents as TFE and likely bacterial membranes are a target of this peptide. To date PDL440-65 is the first antimicrobial peptide identified in a type 1 RIP., (Copyright © 2015 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.)

Ribosome-inactivating proteins are plant cytotoxic enzymes, also present in fungi, algae and bacteria, mainly known for their ability to inhibit protein synthesis. We previously purified and structurally characterized three type 1 RIPs (PD-S1-3) from Phytolacca dioica seeds and four type 1 RIPs (PD-L1-4) from adult plant leaves. Two additional RIPs, named dioicin 1 and dioicin 2, were isolated from leaves of young plants and developing leaves of adult plants. The evidence that P. dioica synthesizes and accumulates these RIPs isoforms suggests that these proteins have been conserved during evolution. Though several aspects of P. dioica type 1 RIP characterization have been studied, some important questions remain to be answered especially with respect to Phytolaccaceae RIP evolution. One of the major problems encountered in approaching RIPs phylogeny concerns the availability of their sequences. In this study, we report the characterization of biological and structural properties of dioicin 1, including the determination of its primary structure by using a combined approach based on Edman degradation, de novo sequencing by ESI-Q-TOF-MS/MS and peptide mapping by MALDI-TOF MS. Knowledge of dioicin 1 primary structure provide us a mean to deepen Phytolaccaceae's RIPs phylogeny. We speculate that both dioicins 1 and 2 share common ancestors with PAP-II and PAP icos-II and that dioicin 1 is not closely related to other members of this clade, thus shedding lights on evolutionary relationships among type 1 RIPs from Phytolaccaceae., (Copyright © 2015 Elsevier Inc. All rights reserved.)

This work examines the crude methanolic extracts of three medicinally important plants native to Pakistan for potent phytotoxic activities and important phytochemicals. These plants include Euphorbia wallichii, Bergenia ciliata and Phytolacca latbenia. The phytotoxic effects were checked at 10,000, 1000, and 100 µg/ml against two economically important standard target species, Triticum aestivum (monocot representative) and Brassica napus (dicot representative). The phytotoxicity effects on seed germination, seedling growth and seedling weight were checked. A simple, cost-effective in vitro phytotoxicity assay (that uses petri plates) was used to evaluate the allelopathic properties of crude extracts. At highest concentration, extracts from all the three plants showed phytotoxic activities such that P. latbenia > E. wallichii > B. ciliata. In seedling growth, root length was affected more than shoot length, whereas among the target species B. napus was found to be more sensitive towards extracts when compared with T. aestivum. Phytochemical analysis showed that P. latbenia is rich in saponins and terpenoids, while E. wallichii and B. ciliata are rich in tannins, terpenoids and cardiac glycoside. P. latbenia also carries a moderate amount of cardiac glycosides., (© The Author(s) 2012.)

We isolated a triterpenoid from an ethanolic extract of Phytolacca decandra and nanoencapsulated it with biodegradable nontoxic polymers of poly(lactide-co-glycolide) to examine if the nanoform of this hitherto unexplored betulinic-acid derivative (NdBA) could produce a stronger anticancer effect by rendering better drug bioavailability and targeted delivery than the nonencapsulated betulinic-acid derivative (dBA). The nanoparticles were characterized with the help of physicochemical and morphological studies involving dynamic light scattering and atomic force microscopy. A549 cancer cells were exposed to NdBA and dBA at the IC50 doses of 50 μg/mL and 100 μg/mL, respectively. Mitochondrial dysfunction-mediated apoptosis was determined by examining the changes in the intracellular calcium content, the reactive oxygen species accumulation, the cytochrome c release, the upregulation of Bcl-2-associated-X protein (Bax) and caspase 3, the downregulation of B cell lymphoma 2, and the mitochondrial membrane potential (ΔΨm) depolarization. Apoptosis was also verified by acridine orange staining observed under fluorescence microscopy and annexin V-fluorescein isothiocyanate/propidium iodide staining through flow cytometric studies. The levels of intracellular adenosine triphosphate/adenosine diphosphate ratio decreased, and the ATPase activity increased more strikingly in A549 cells exposed to NdBA than in A549 cells exposed to dBA. Overall results showed that both drugs directly target the mitochondrial oxidative phosphorylation system, with NdBA having a stronger effect, indicating NdBA to be a better candidate for the development of an anticancer drug for use against lung adenocarcinomas., (Copyright © 2013. Published by Elsevier B.V.)

Objective: Oleanolic acid (OA) has been reported to have anticancer effects, but the extent of its cytotoxicity, its ability to interact with nuclear DNA, its action against skin melanoma, as well as the molecular mechanism of its action against cell proliferation and in support of cell death are still unexplored. This led us to examine the efficacy of OA, a bioactive compound isolated from Phytolacca decandra, on these issues in the present investigation., Methods: Studies related to analyses of cell viability, drug-DNA interaction, cell proliferation, cell cycle and epidermal growth factor receptor (EGFR) activity were performed. To investigate whether cells undergo apoptosis, studies like fluorescence microscopy, poly (ADP-ribose) polymerase (PARP) degradation, annexin V-fluorescein isothiocyanate/propidium iodide assay, alteration in mitochondrial membrane potential and activity of some relevant signaling proteins were performed., Results: OA displayed a minimal and negligible cytotoxic effect on normal HaCaT cells (skin keratinocytes) and peripheral blood mononuclear cells but by contrast it reduced A375 cell viability significantly. OA interacted with nuclear DNA quickly after exposure. It acted as an anti-proliferative agent. It suppressed EGFR activity. OA administration led the cells to mitochondria-dependent caspase 3-mediated apoptosis., Conclusion: OA interacts with cellular DNA, inhibits proliferation possibly through modulating EGFR activity and induces mitochondria-dependent caspase 3-mediated apoptosis in A375 cells which would qualify it as a potent anticancer agent.

Objective: To extract and separate toxic components from Phytolaccae Radix, and to comare the changes in toxicity of Phytolaccae Radix before and after being processed with vinegar., Method: The mucous membrane irritation response, mouse peritoneal inflammation model and in vitro macrophages release NO model were applied to compared the changes in inflammatory toxicity of toxic components from Phytolaccae Radix before and after being processed with vinegar., Result: Toxic components of Phytolacca Radix had significant inflammatory toxicity, which could cause conjunctival edema in rabbits, and increase of PGE2 and macrophages release NO content in peritoneal exudate in mice. After being processed with vinegar, they showed reduced irritation, which resulted in decrease of PGE2 and macrophages release NO content in peritoneal exudate in mice., Conclusion: After being processed with vinegar, the toxicity of toxic components from Phytolacca Radix decreased obviously.

Objective: Preventive measures against skin melanoma like chemotherapy are useful but suffer from chronic side effects and drug resistance. Ethanolic extract of Phytolacca decandra (PD), used in homeopathy for the treatment of various ailments like chronic rheumatism, regular conjunctivitis, psoriasis, and in some skin diseases was tested for its possible anticancer potential., Methods: Cytotoxicity of the drug was tested by conducting 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay on both normal (peripheral blood mononuclear cells) and A375 cells. Fluorescence microscopic study of 4',6-diamidino-2-phenylindole dihydrochloride-stained cells was conducted for DNA fragmentation assay, and changes in cellular morphology, if any, were also recorded. Lactate dehydrogenase activity assay was done to evaluate the percentages of apoptosis and necrosis. Reactive oxygen species (ROS) accumulation, if any, and expression study of apoptotic genes also were evaluated to pin-point the actual events of apoptosis., Results: Results showed that PD administration caused a remarkable reduction in proliferation of A375 cells, without showing much cytotoxicity on peripheral blood mononuclear cells. Generation of ROS and DNA damage, which made the cancer cells prone to apoptosis, were found to be enhanced in PD-treated cells. These results were duly supported by the analytical data on expression of different cellular and nuclear proteins, as for example, by down-regulation of Akt and Bcl-2, up-regulation of p53, Bax and caspase 3, and an increase in number of cell deaths by apoptosis in A375 cells., Conclusion: Overall results demonstrate anticancer potentials of PD on A375 cells through activation of caspase-mediated signaling and ROS generation.

The in vivo anthelmintic (AH) activity of the ethanolic extract from leaves of Phytolacca icosandra was evaluated in goats artificially infected with Haemonchus contortus. Parasite naïve goats were artificially infected with 3000 H. contortus infective larvae per animal. Once the infection was patent (day 28 post-infection) all the animals were sampled to determine the faecal egg counts (FEC) for five consecutive days. Two groups of animals were formed balanced for their FEC and body-weight (BW) (n=6/group): the non-treated control group and the treated group in which goats were individually administered with the ethanolic extract of P. icosandra. The extract was administered orally using gelatin capsules (250 mg/kg BW) which were dosed on two consecutive days using a pill-dispenser. Faecal samples were collected from each animal from the day of dosage (Day 0) on a daily basis to determine the number of eggs per gram of feces (EPG) for 15 days post-treatment (PT). The FEC of the two groups were compared using the repeated measures analyses of variance using the log transformed data Ln (FEC+1). The presence of saponins, coumarins, flavonoids, steroids and terpenoids were detected by standard methodologies in the extract. The P. icosandra ethanolic extract was further analyzed by gas chromatography (GC) coupled to a mass spectrometry (GC-MS). A significant reduction in FEC was observed in the treated group compared to the control from day 7 until day 15 PT (P<0.05). The highest percentage reduction (72%) was found on day 11 PT. No adverse reactions were observed in all treated animals for the entire trial. The GC-MS analysis of the organic extracts revealed the presence of three fatty acids as compounds with highest abundance. The three compounds that were identified by their mass fragmentation patterns were: 2-Pentadecanone, 6, 10, 14-trimethyl (RT 10.3 min), Pentadecanoic acid, 14-methyl-, methyl ester (RT 10.8 min) and Hexadecanoic acid, ethyl ester (RT 11.2 min). It is concluded that the P. icosandra ethanolic extract obtained from leaves showed in vivo anthelmintic activity against H. contortus when administered orally to goats at a dose of 250 mg/kg BW on two consecutive days. The dose used did not cause any negative effects on the health of goats., (Copyright © 2012. Published by Elsevier B.V.)

Objective: To examine if a homeopathic mother tincture (Phytolacca Decandra) is capable of precipitating silver nanoparticles from silver nitrate (AgNO(3)) and to characterize the biosynthesized nanoparticles for evaluating their biological activities., Methods: A total of 100 mg of AgNO(3) was added to 20mL of Milli-Q water and stirred vigorously. Then 5mL of the homeopathic mother tincture of Phytolacca Decandra (ethanolic root extract of Phytolacca decandra) was added and stirred continuously. Reduction took place rapidly at 300K and completed in 10 min as shown by stable light greenish-yellow color of the solution which gave colloid of silver nanoparticles. The colloid solution was then centrifuged at 5000×g to separate the nanoparticles for further use. The nanoparticles were characterized by spectroscopic analysis, particle size analysis and zeta potential measurements, and morphology was analyzed by atomic force microscopy. The drug-DNA interaction was determined by circular dichroism spectrophotometry and melting temperature profiles by using calf thymus DNA as the target. The biological activities were determined using a cancer cell line A549 in vitro and using bacteria Escherichia coli and fungus Saccharomyces cerevisiae as test models., Results: Phytolacca Decandra precipitated silver nanoparticles in ambient conditions. The nanoparticles had 91 nm particle size, with polydispersity index of 0.119 and zeta potential of -15.6 mV. The silver nanoparticles showed anticancer and antibacterial properties, but no clear antifungal properties., Conclusion: This could be a novel environment-friendly method to biosynthesize silver nanoparticles using a cost-effective, nontoxic manner. The homeopathic mother tincture may utilize this property of nano-precipitation in curing diseases or disease symptoms.

A new triterpenoid saponin, named esculentoside T (1), together with five known compounds (2-5), was isolated from the roots of Phytolacca acinosa. The structures of these compounds were elucidated on the basis of spectral data.

A series of 46 compounds derived from esculentoside A and its aglycone were synthesized and characterized. The effect of these compounds on lipopolysaccharide (LPS)-induced NO production, haemolytic activity, and cell viability was evaluated. Structure-activity relationship was established by comparing the derivatives of esculentoside A with its aglycone derivatives. Both the aglycone and its derivatives showed higher inhibitory effects on LPS-induced NO production, and lower haemolytic activities than esculentoside A and its derivatives., (Copyright © 2011 Verlag Helvetica Chimica Acta AG, Zürich.)

The development of anthelmintic resistance has impacted on the success of conventional anthelmintics (AH) for the control of gastrointestinal nematodes in grazing/browsing sheep and goats. Medicinal plants from the traditional herbolary in Mexico may provide new candidates that can be explored as alternative sources of AHs for ruminants. This study evaluated the leaf extracts derived from Phytolacca icosandra against infective L(3) larvae and eggs from Haemonchus contortus collected from sheep. Three extracts of different polarities were obtained from the leaf plants using ethanol, n-hexane and dichloromethane as the solvents. The effectiveness of the in vitro AH activity of the plant extracts was evaluated using larval migration inhibition (LMI) and egg hatch (EHA) assays. For the LMI assays, the ethanolic extract of P. icosandra showed 55.4% inhibition of larval migration at 2mg/mL (p<0.05). The dichloromethane extract of P. icosandra showed 67.1% inhibition of migration at 3mg/mL (p<0.05) and a dose-dependent response with an LD(50) of 0.90 mg/mL. The n-hexane extract failed to show inhibition of larval migration at any concentration explored. In the EHA for the ethanol extract, the lowest concentration tested (0.15 mg/mL) resulted in inhibition of egg hatching greater than 72.6%. Therefore, the LD(50) could not be calculated for this extract. The LD(50) of the dichloromethane extract of P. icosandra was 0. 28 mg/mL. An egg hatch inhibition greater than 90% was observed with both the ethanolic and dichloromethane extracts when using a concentration of 0.90 mg/mL or higher. The n-hexane extract failed to show egg hatch inhibition at any concentration tested. The AH activity reported for P. icosandra could be attributable to the flavonoids, steroids, terpenoids, coumarins and/or saponins that were present in the ethanolic and dichloromethane extracts. A combination of more than one component may also explain the observed AH activity against the H. contortus life stages that were evaluated. In conclusion, the ethanolic and dichloromethane extracts of P. icosandra showed clear in vitro AH activity against the H. contortus eggs and the L(3) larvae. However, the hexanic extract of the plant leaves failed to show any in vitro AH activity., (Copyright © 2011 Elsevier B.V. All rights reserved.)

Objective: To study the changes of mucosa irritation of crude and processed Radix Phytolaccae., Method: Gastrointestinal experiments on rats and mice and the rabbits' eyes irritation experiments were applied to investigate the changes of mucosa irritation of crude and processed Radix Phytolaccae., Result: The mucosa irritation of processed Radix Phytolaccae decreased obviously compared to crude one., Conclusion: After being processed with vinegar, the mucosa irritation on Radix Phytolaccae decreased obviously.

A saponin-rich extract of Phytolacca dioica L. berries, its acid hydrolysate, and its major aglycone, phytolaccagenin, were assayed for antifungal activity against ATCC standard cultures of Candida albicans and Cryptococcus neoformans, and against clinical isolates of these fungi. The activity of the extract was either low or negligible, but the hydrolysate, containing the sapogenins, including phytolaccagenin, and also pure phytolaccagenin, showed promising antifungal potency. Hydrolysis of a natural product extract is shown to be a useful modification leading to improved bioactivity.

Four known serjanic acid glycosides were isolated from the fruits of Phytolacca rugosa and characterized mainly by 2D NMR spectroscopy and mass spectrometry. This aglycon has a chemotaxonomic significance for the genus Phytolacca.

The use of ultra-diluted natural products in the management of disease and treatment of cancer has generated a lot of interest and controversy. We conducted an in vitro study to determine if products prescribed by a clinic in India have any effect on breast cancer cell lines. We studied four ultra-diluted remedies (Carcinosin, Phytolacca, Conium and Thuja) against two human breast adenocarcinoma cell lines (MCF-7 and MDA-MB-231) and a cell line derived from immortalized normal human mammary epithelial cells (HMLE). The remedies exerted preferential cytotoxic effects against the two breast cancer cell lines, causing cell cycle delay/arrest and apoptosis. These effects were accompanied by altered expression of the cell cycle regulatory proteins, including downregulation of phosphorylated Rb and upregulation of the CDK inhibitor p27, which were likely responsible for the cell cycle delay/arrest as well as induction of the apoptotic cascade that manifested in the activation of caspase 7 and cleavage of PARP in the treated cells. The findings demonstrate biological activity of these natural products when presented at ultra-diluted doses. Further in-depth studies with additional cell lines and animal models are warranted to explore the clinical applicability of these agents.

Introduction: The lack of pharmacopoeial methodologies for the quality control of plants used for therapeutic purposes is a huge problem that impacts directly upon public health. In the case of saponins, their great structural complexity, weak glycoside bonds and high polarity hinder their identification by conventional techniques., Objective: To apply high-performance liquid chromatography-electrospray tandem mass spectrometry (HPLC-ESI/MS(n)) to identify the O-glycoside sequence of saponins from the roots of Phytolacca bogotensis., Methodology: Saponins were isolated by preparative HPLC and characterised by NMR spectroscopic experiments. Collision-induced dissociation (CID) of isolated saponins was performed producing typical degradation reactions that can be associated with several glycosidic bonds as empirical criteria. A method using solid-phase extraction (SPE) and HPLC/ESI-MS(n) for the characterisation of saponins and identification of novel molecules is described., Results: Three saponins reported for the first time in P. bogotensis were isolated and characterised by NMR spectroscopy. Characteristic cross ring cleavage reactions have been used as empirical criteria for the characterisation of the glycosidic bonds most frequently reported for Phytolacca saponins. One new saponin was proposed on the basis of empirical criteria, and other five saponins were identified for the first time for P. bogotensis using HPLC-ESI/MS(n)., Conclusion: Electrospray ionisation in combination with tandem mass spectrometry has been established as a powerful tool for the profiling of saponins from roots of P. bogotensis. CID proved to be a useful tool for the characterisation and identification of known and novel saponins from the plant family Phytolaccaceae and can be used for quality control purposes of crude plant extracts.

Through the action of glycosyltransferases, a plant can biosynthetically assemble small different aglycons or 'templates' to various polysaccharides to produce numerous glycoconjugates differing in the type of the attached aglycon, the anomeric configuration of C-1 of the glycosylating sugar, the type of sugar and the different position of attachments of the sugar unit present in the polysaccharide chain. The position of attachments and the anomeric configuration of the different sugar present in the polysaccharide create the opportunity to generate molecules with either the same or very close molecular weights, which have relative structural similarity--forming isobaric and positional isomers. Although isomeric differentiation was once considered outside of the domain of mass spectrometry, this task can now be resolved using tandem mass spectrometry. In a standardized purified glycoconjugate fraction (SPT01) from Phytolacca bogotensis, we report conventional electrospray ionization mass spectrometry and collision-induced dissociation (CID) MS/MS parameters which favored the formation of characteristic product ions. This allowed us to suggest the type of sugar linkages present in a specific glycoconjugate. Ten new glycoconjugate are described from this plant and another twelve known saponins were structurally characterized using the automatic MSn acquisition mode. The differentiation of two pairs of positional isomers and four isobaric glycosides and the production of a library of 30 glycosides present in P. bogotensis were accomplished., (Copyright 2009 John Wiley & Sons, Ltd.)

The amino acid sequence and glycan structure of PD-L1, PD-L2 and PD-L3, type 1 ribosome-inactivating proteins isolated from Phytolacca dioica L. leaves, were determined using a combined approach based on peptide mapping, Edman degradation and ESI-Q-TOF MS in precursor ion discovery mode. The comparative analysis of the 261 amino acid residue sequences showed that PD-L1 and PD-L2 have identical primary structure, as it is the case of PD-L3 and PD-L4. Furthermore, the primary structure of PD-Ls 1-2 and PD-Ls 3-4 have 81.6% identity (85.1% similarity). The ESI-Q-TOF MS analysis confirmed that PD-Ls 1-3 were glycosylated at different sites. In particular, PD-L1 contained three glycidic chains with the well known paucidomannosidic structure (Man)(3) (GlcNAc)(2) (Fuc)(1) (Xyl)(1) linked to Asn10, Asn43 and Asn255. PD-L2 was glycosylated at Asn10 and Asn43, and PD-L3 was glycosylated only at Asn10. PD-L4 was confirmed to be not glycosylated. Despite an overall high structural similarity, the comparative modeling of PD-L1, PD-L2, PD-L3 and PD-L4 has shown potential influences of the glycidic chains on their adenine polynucleotide glycosylase activity on different substrates.

Phytolacacinoside A (1), a novel triterpenoid saponin, together with the seven known compounds, was isolated from 75% ethanol extract of the root of Phytolacca acinosa Roxb (Phytolaccaceae). Their structures were elucidated on the basis of analysis of spectroscopic data and physicochemical properties as 3-O-beta-[(beta-d-glucopyranosyl-(1 --> 4)-O-beta-D-xylopyranosyl)]-11beta-methoxy-jaligonic acid 30-methyl ester 28-O-beta-D-glucopyranoside (1), 3-O-beta-[(beta-D-glucopyranosyl-(1 --> 4)-O-beta-D-xylopyranosyl)]-jaligonic acid 30-methyl ester 28-O-beta-D-glucopyranoside (2, esculentoside G), 3-O-beta-[(beta-D-glucopyranosyl-(1 --> 4)-O-beta-D-xylopyranosyl)]-jaligonic acid 30-methyl ester (3, phytolaccoside E), 3-O-beta-D-xylopyranosyl-jaligonic acid 30-methyl ester (4, phytolaccoside B), hypaphorine (5), palmitic acid monoglyceride (6), beta-sitosterol (7), and daucosterol (8).

Phytolaccoside B (1), an antifungal monodesmoside triterpenoid glycoside isolated from berries of Phytolacca tetramera Hauman (Phytolaccaceae), alters the morphology of yeasts and molds. The malformations were similar to those produced by enfumafungin, a known inhibitor of (1-->3)-beta-D-glucan synthase, an enzyme that catalyzes the synthesis of (1-->3)-beta-D-glucan, one of the major polymers of the fungal cell wall. However, enzymatic assays revealed that 1 did not inhibit (1-->3)-beta-D-glucan synthase, but it did produce a notable enhancement of the chitin synthase 1 activity and, concomitantly, a rise in chitin, another important polymer of the fungal cell walls. This finding was corroborated by fluorescence microscopy and also by quantification of the chitin. In addition, a 2-fold increase in the thickness of the fungal cell wall was observed with transmission electronic microscopy. On the other hand, 1 neither bound to ergosterol nor caused hemolysis of red blood cells, although some fungal membrane damage was observed at the MIC of 1.

Seeds from Phytolacca dioica L. contain at least three N-glycosylated PD-Ss, type 1 ribosome-inactivating proteins (RIPs), which were separated and purified to homogeneity by conventional chromatographic techniques. ESI-Q-TOF mass spectrometry provided the accurate M(r) of native PD-S1 and PD-S3 (30957.1 and 29785.1, respectively) and the major form PD-S2 (30753.8). As the amino acid sequence of PD-S2 was already known, its disulfide pairing was determined and found to be Cys34-Cys262 and Cys88-Cys110. Further structural characterization of PD-S1 and PD-S3 (N-terminal sequence determination up to residue 30, amino acid analysis and tryptic peptide mapping) showed that the three PD-Ss shared the entire protein sequence. To explain the different chromatographic behaviour, their glycosylation patterns were characterized by a fast and sensitive mass spectrometry-based approach, applying a precursor ion discovery mode on a Q-TOF mass spectrometer. A standard plant paucidomannosidic N-glycosylation pattern [Hex(3), HexNAc(2), deoxyhexose(1), pentose(1)] was found for PD-S1 and PD-S2 on Asn120. Furthermore, a glycosylation site carrying only a HexNAc residue was identified on Asn112 in PD-S1 and PD-S3. Finally, considering the two disulfide bridges and the glycan moieties, the experimental M(r) values were in agreement with the mass values calculated from the primary structure. The complete characterization of PD-Ss shows the high potential of mass spectrometry to rapidly characterize proteins, widespread in eukaryotes, differing only in their glycosylation motifs.

Multiple sequence alignment analysis of ribosome inactivating proteins (RIPs) has revealed the occurrence of an invariant seryl residue in proximity of the catalytic tryptophan. The involvement of this seryl residue in the catalytic mechanism of RIPs was investigated by site-directed mutagenesis in PD-L4, type 1 RIP isolated from Phytolacca dioica leaves. We show that the replacement of Ser211 with Ala apparently does not influence the N-beta-glycosidase activity on ribosomes (determined as IC(50) in a cell-free system), but it reduces the adenine polynucleotide glycosylase activity (APG), assayed spectrophotometrically on other substrates such as DNA, rRNA, and poly(A). The ability of PD-L4 to deadenylate polynucleotides appears more sensitive to the Ser211Ala replacement when poly(A) is used as substrate, as only 33% activity is retained by the mutant, while with more complex and heterogeneous substrates such as DNA and rRNA, its APG activity is 73% and 66%, respectively. While the mutated protein shows a conserved secondary structure by CD, it also exhibits a remarkably enhanced tryptophan fluorescence. This indicates that, although the overall protein tridimensional structure is maintained, removal of the hydroxyl group locally affects the environment of a Trp residue. Modelling and docking analyses confirm the interaction between Ser211 and Trp207, which is located within the active site, thus affecting RIP adenine polynucleotide glycosylase activity. Data accumulated so far confirm the potential involvement of Ser211 in the catalytic mechanism of type 1 RIP PD-L4 and a possible role in stabilizing the conformation of Trp207 side chain, which participates actively in the protein enzymatic activity., ((c) 2007 Wiley-Liss, Inc.)

PD-L1 is a highly glycosylated type 1 ribosome inactivating protein, from Phytolacca dioica leaves, with the peculiarity to act also as a DNase. PD-L1 has been successfully crystallized using vapour diffusion and seeding techniques. Crystals belong to the monoclinic C2 space group, with unit cell dimensions a=161.01, b=34.73, c=120.63 A, beta=127.99 degrees . Two molecules are present in the asymmetric unit. Phase determination has been achieved using molecular replacement.

PD-L4, a type 1 ribosome inactivating protein from Phytolacca dioica leaves, has been successfully crystallized using vapour diffusion methods and PEG 4000 as a precipitant agent. In addition, crystals of a PD-L4 mutant, which has been recently observed to have a lower polynucleotide-adenosine glycosidase activity on DNA, rRNA and poly (A) substrates, have been obtained. To gather information on PD-L4 reaction mechanism both forms have been co-crystallized with adenine, the major product of their catalytic reaction. Diffraction patterns extend to atomic resolution and crystals belong to the orthorhombic P2(1)2(1)2(1) space group, with one molecule in the asymmetric unit. Structure determination has been achieved using molecular replacement; preliminary electron density maps have clearly given evidence of adenine binding.

Phytolacca acinosa Roxb. (P. acinosa) is a recently discovered manganese hyperaccumulator plant from southern China. It is a good candidate for phytoremediation of manganese(Mn) polluted soil for its high biomass and fast growth. Knowledge of the tissue localization and identification of heavy metals can provide essential information on metal toxicity and bioaccumulation mechanisms. Synchrotron radiation X-ray fluorescence spectroscopy (SRXRF) microprobe was used in this study to investigate the cellular distributions of Mn and other elements in root, stem, leaf, petiole and midrib of P. acinosa. The highest Mn content was found in the vascular tissues of root, stem, petiole and midrib. Cortex in root played a key role in Mn absorption and Mn was limited in the vascular bundle during the process of transportation in stem. Moreover, Mn content in leaf epidermis was higher than that in mesophyll, which suggested that the sequestration of Mn in leaf epidermis might be one of the detoxification mechanisms of P. acinosa. The significance of other elemental (such as P, S, K, Ca, Fe, Zn and Cu) distribution patterns and the correlation with Mn were also discussed.

Pokeweed antiviral protein (PAP) is a ribosome-inactivating protein isolated from the pokeweed plant (Phytolacca americana) that inhibits the proliferation of several plant and animal viruses. We have shown previously that PAP and nontoxic mutants of PAP can directly depurinate brome mosaic virus (BMV) RNA in vitro, resulting in reduced viral protein translation. Here we expand on these initial studies and, using a barley protoplast system, demonstrate that recombinant PAP and nontoxic mutants isolated from E. coli are able to reduce the accumulation of BMV RNAs in vivo. Pretreatment of only BMV RNA3 with PAP prior to transfection of barley protoplasts reduced the accumulation of all BMV RNAs, with a more severe effect on subgenomic RNA4 levels. Using in vitro RNA synthesis assays, we show that a depurinated template causes the BMV replicase to stall at the template nucleotide adjacent to the missing base. These results provide new insight into the antiviral mechanism of PAP, namely that PAP depurination of BMV RNA impedes both RNA replication and subgenomic RNA transcription. These novel activities are distinct from the PAP-induced reduction of viral RNA translation and represent new targets for the inhibition of viral infection.

Objective: To make an attempt at the multi-element speciation in the Chinese medicinal herbs by determining the concentrations of 25 elements in different extraction solutions., Method: Firstly, five Chinese medicinal herbs (Buddleja officinalis, Dictamnus dasycarpus, Myristica fragrans, Albizia judibrissin and Inula japonica) from the same region of China were treated to obtain water-soluble phase, lipid-soluble phase and non-soluble phase by water extraction, organic solvent extraction and acid digestion, respectively. Secondly, Phytolacca acinosa, a Chinese medicinal herb collected from 9 regions of China, was extracted by 0% EtOH, 50% EtOH, 75% EtOH, 95% EtOH, respectively, referring the Chinese Pharmacopoeia. Finally, the concentrations of 25 elements, such as Be, Cr, Cu, Zn, Ge, Sr, Y, Mo, Cd, Tl, Pb and REEs, in the above three phases were determined by ICP-MS., Result: Under the optimal conditions, all the 25 elements could be determined with detection limits ranged from 0.003 to 0.71 ng x g(-1). The average recoveries of the elements in P. acinosa were 88% approximately 119%, with the relative standard deviations 1.7% approximately 13.3%. It was observed that the determined 25 elements distributed in all the water-soluble, lipid-soluble and non-soluble phases, indicating that the inorganic species, organicspecies, as well as the protein bound species were coexisted in the herbs. Big differences of the element extraction rates could be found by using different ethanol solutions., Conclusion: With the aid of the obtained results, we may increase the extraction of necessary elements while decrease that of the toxic elements from the herbs by choosing a suitable solvent during the drug production.